Immortalized egg shell gland epithelial cell line and application thereof

Abstract

The invention discloses an immortalized egg shell gland epithelial cell line and application thereof. The invention belongs to the technical field of biology, and particularly relates to an immortalized egg shell gland epithelial cell line and application thereof. The construction method of the immortalized egg shell gland epithelial cell line comprises the following steps: 1) obtaining SV40 overexpression lentivirus; 2) transfecting the lentivirus obtained in the step 1) to chicken primary eggshell gland epithelial cells; amplifying the transfected cells for 3-4 generations, and screening puromycin to obtain successfully transfected eggshell gland epithelial cells; and (3) carrying out subculture on the successfully transfected eggshell gland epithelial cells obtained in the step (2) to obtain the stably subcultured immortalized eggshell gland epithelial cells. The immortalized egg shell gland epithelial cell provided by the invention can provide a reliable cell model for researching related functions of poultry egg shell glands, and has an important value for developing a novel feed additive capable of improving chicken genital tract health and egg shell quality.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an immortalized eggshell gland epithelial cell line and its applications. Background Technology

[0002] Egg formation involves the sequential processes of the infundibulum, bulge, isthmus, shell gland, and vagina of the hen's oviduct, taking approximately 24 hours. The formation and mineralization of the eggshell in the shell gland alone takes about 20 hours, making it the most crucial and longest stage of egg production. When the shell gland is infected by exogenous pathogens or suffers inflammatory damage due to aging, the eggshell thickness and strength significantly decrease, and the eggshell breakage rate significantly increases. Current research on the mechanisms regulating shell gland function can only be conducted on primary chicken shell gland epithelial cells. However, primary cells are highly differentiated, short-lived, and have weak proliferative capacity; they exhibit natural aging after 72 hours of culture, and their specific functions rapidly decline, failing to meet the requirements for multiple passages in cell experiments.

[0003] Establishing eggshell gland epithelial cell lines has become a key pathway to overcome current research bottlenecks. This important technology will provide stable, passaged cell material, significantly improving the accuracy and reproducibility of research results and providing favorable conditions for in-depth analysis of eggshell biomineralization mechanisms and the regulatory roles of nutritional factors. Simultaneously, it effectively reduces reliance on live experimental animals, lowering experimental costs. Currently, there are no reported methods or studies on immortalized chicken eggshell gland epithelial cells. Therefore, establishing immortalized chicken eggshell gland epithelial cells is of significant value for research on poultry reproductive physiology, elucidation of eggshell formation mechanisms, and the development of prevention and control strategies for reproductive tract diseases. Summary of the Invention

[0004] The main problem to be solved by this invention is how to establish immortalized epithelial cells of chicken shell glands.

[0005] To address the aforementioned problems, this invention provides a method for constructing an immortalized eggshell gland epithelial cell line.

[0006] This invention first provides a method for constructing an immortalized eggshell gland epithelial cell line, comprising the following steps: 1) Obtaining SV40 overexpression lentivirus; 2) Transfect the lentivirus obtained in step 1) into primary chicken shell gland epithelial cells; after the transfected cells are expanded for 3-4 generations, they are screened for successful transfection with puromycin to obtain shell gland epithelial cells. 3) Passage the successfully transfected eggshell gland epithelial cells obtained in step 2) to obtain stable, immortalized eggshell gland epithelial cells.

[0007] Further, the structure of the SV40 overexpression lentiviral vector described in step 1) is as follows: it is a recombinant vector obtained by inserting a DNA fragment with the sequence SEQ ID No:1 between the EcoRI and XbaI restriction sites of the starting vector pLVX-EF1α-IRES-puro, while keeping the other sequences of the vector pLVX-EF1α-IRES-puro unchanged.

[0008] Further, in step 2), the puromycin screening concentration is 1 μg / mL, and the treatment time is 2 days.

[0009] Furthermore, the subculturing described in step 3) is for 15 generations.

[0010] The present invention also provides a method for constructing an inflammation model of immortalized eggshell gland epithelial cells, wherein the immortalized eggshell gland epithelial cells described above are added to a culture medium containing LPS for culture to obtain the inflammation model.

[0011] Furthermore, the concentration of LPS used is 10 µg / mL.

[0012] This invention also provides the application of the method described above in the preparation of immortalized eggshell gland epithelial cell lines.

[0013] This invention also provides the application of the method described above in the preparation of products containing immortalized eggshell gland epithelial cell lines.

[0014] The present invention also provides an immortalized chicken uterine epithelial cell line obtained by the method described above.

[0015] In one specific embodiment, the chicken uterine epithelial immortalized cell line is the chicken uterine epithelial immortalized cell line iCUECs2025, which was deposited on November 4, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46710.

[0016] This invention also provides the application of the immortalized eggshell gland epithelial cells described above in the study of eggshell gland-related functions in poultry and / or the screening of novel feed additives to improve eggshell quality.

[0017] This invention discloses a method for immortalizing chicken shell gland epithelial cells, which overcomes the limitation of primary chicken shell gland epithelial cells not being able to be passaged multiple times continuously. This method plays an important role in research on inflammation and immunity in the reproductive tract of laying hens and the regulation of eggshell quality. Specifically, this invention involves constructing a vector overexpressing SV40 lentivirus to transfect primary chicken shell gland epithelial cells. Then, appropriate concentrations of puromycin are used to screen the transfected cells, successfully obtaining a chicken shell gland epithelial cell line that can be passaged multiple times and exhibits good growth (accession number: CGMCC No. 46710). Experiments have confirmed that this shell gland epithelial cell line maintains morphological stability up to 15 passages and can be used to construct cellular inflammation models. Based on its stable passage characteristics and model application value, the immortalized chicken shell gland epithelial cells provided by this invention offer a reliable cell model for studying the functions related to the chicken shell gland and are of significant value for developing novel feed additives that can improve the health of the chicken reproductive tract and eggshell quality.

[0018] Preservation Instructions Scientific Description: Immortalized Chicken Uterine Epithelial Cell Line Referenced biological material (strain): iCUECs2025 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Collection institution abbreviation: CGMCC Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: November 4, 2025 Registered with the China National Collection Center (CGMCC) No. 46710. Attached Figure Description

[0019] Figure 1 This is an immunofluorescence identification image of chicken primary eggshell gland epithelial cells according to the present invention.

[0020] Figure 2 This is a schematic diagram of the SV40 overexpression lentiviral vector structure of the present invention.

[0021] Figure 3 This is a diagram of cells transfected with SV40 overexpressing lentivirus according to the present invention.

[0022] Figure 4 This is a diagram of immortalized eggshell gland epithelial cells expanded to the 15th generation according to the present invention.

[0023] Figure 5 This is an immunofluorescence identification image of immortalized eggshell gland epithelial cells according to the present invention.

[0024] Figure 6This is a diagram showing the SV40 expression in primary and immortalized eggshell gland epithelial cells of the present invention. Wherein, M represents the marker; 1 represents primary chicken eggshell gland epithelial cells; and 2 represents immortalized eggshell gland epithelial cells.

[0025] Figure 7 The cell activity and nitric oxide content of the immortalized eggshell gland epithelial cell inflammation model of the present invention are shown. Detailed Implementation

[0026] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0028] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0029] The 35-week-old Hy-Line Brown laying hens used in the following examples are described in: Jianmin Zhou, Yu Fu, Guanghai Qi, Jinjun Dai, Haijun Zhang, Jing Wang, Shugeng Wu. Yeast cell-wall polysaccharides improve immunity and attenuate inflammatory response via modulating gut microbiota in LPS-challenged laying hens. International Journal of Biological Macromolecules. 2023 Jan 1;224:407-421. doi: 10.1016 / j.ijbiomac.2022.10.133. This biological material is available to the public from the applicant and is intended solely for the purpose of replicating experiments of this invention and shall not be used for any other purpose.

[0030] The main reagent information in the following examples is as follows: fetal bovine serum (Gibco, 1414426), complete epithelial cell culture medium (Cybercare, Primed-icell-001), 0.25% trypsin (Gibco, 1734858), collagenase I (Gibco, 17018029), paraformaldehyde (Solarbio, P1110), DAPI (Solarbio, C0060), CK18 primary antibody (Bioss, BS-2043R), CoraLite488-labeled goat anti-rabbit secondary antibody (Proteintech, SA00013-2), Fluoromount-G fluorescent mounting medium (Sourthern Biotech, 0100-01), animal tissue / cell genomic DNA extraction kit (Solarbio, D1700), D2000 DNA Ladder (Solarbio, M1060), puromycin (Solarbio, P8230), lipopolysaccharide (Sigma, L4391), Nitric Oxide Kit (Beyotime, S0021S).

[0031] The vector pLVX-EF1α-IRES-puro used in the following examples was purchased from Beijing Bio-Bio Biotechnology Co., Ltd. (bio-118231).

[0032] The following examples used statistical software to process the data. The experimental results are expressed as mean ± standard deviation, and Student's T test was used. P <0.05 (*) indicates a significant difference. P <0.01 (**) indicates a highly significant difference. P <0.001 (***) indicates a highly significant difference.

[0033] Example 1: Construction of an immortalized eggshell gland epithelial cell line 1. Isolation, culture, and immunofluorescence identification of primary chicken eggshell gland epithelial cells Chickens were euthanized by exsanguination via the carotid artery. Eggshell gland tissue was harvested in a laminar flow hood and placed in PBS buffer containing penicillin-streptomycin. The connective tissue surrounding the eggshell gland was removed, and the tissue was washed three times with PBS buffer. The tissue was then immersed in 75% ethanol for 2 minutes and washed with PBS buffer. The eggshell gland horn was cut along its longitudinal axis to expose the internal cavity. The inner membrane of the eggshell gland was separated using ophthalmic scissors and forceps, and rinsed in PBS buffer until the liquid was clear. The tissue was digested with 0.1% type I collagenase at 3 times its volume in a shaking water bath at 37°C for 1-1.5 hours. The digested tissue mixture was passed through a 100μm sieve, and the filtrate was collected. The mixture was centrifuged at 1500 rpm for 10 minutes, and the supernatant was discarded. The pellet was resuspended in complete epithelial cell culture medium and transferred to a T-25 culture flask. The flask was incubated at 37°C in a 5% CO2 incubator, with the medium changed every 24 hours.

[0034] Then, the isolated chicken primary eggshell gland epithelial cells were identified by immunofluorescence. The specific steps are as follows: Cell slides: Place 3 glass slides in a 24-well plate, add 1 mL of culture medium to each well, and add 2 × 10⁶ cells. 4 One per well. Incubate for 2 hours or overnight.

[0035] Fixation: After cell crawling, aspirate the culture medium, wash once with PBS buffer, add 4% PFA and fix at 4℃ for 30 min. Wash with PBS buffer 3 times for 5 min each time.

[0036] Membrane disruption and blocking: Remove moisture from the glass slide and place it on the culture dish support. Then prepare the glass slide blocking solution by mixing 0.5% Tritium X-100 with PBS at a 1:1 ratio and adding 10% serum. Take 50 μL of the membrane disruption and blocking solution and drop it onto the waterproof membrane. Cover the glass slide with the side containing cells for 2 hours.

[0037] Primary antibody incubation: Take 50uL of cytokeratin 18 (CK18) antibody (diluted with PBS 1:100) onto a waterproof membrane, cover with a glass slide containing cells and store at 4℃.

[0038] Secondary antibody incubation: Incubate the secondary antibody (diluted with PBS 1:500) at room temperature in the dark for 2 hours, then wash with PBS 3×5 min / time, stain with DAPI (DAPI:PBS=1:1000) for 5 min, and wash with PBS 3×5 min / time.

[0039] Embedding: Place 1 drop of Fluoromount-G on each slide and cover the side with cells.

[0040] Observation: Observe and take pictures using a fluorescence microscope.

[0041] CK18 is an intermediate fibrous protein that forms part of the epithelial cell cytoskeleton and is commonly used as an epithelial cell-specific marker in immunofluorescence detection. Figure 1 It can be seen that the CK18 protein is positively expressed in the isolated cell nucleus, indicating that the cell is an eggshell gland epithelial cell. The positive expression rate of the CK18 gene is 97%, which can be used for subsequent immortalization operations.

[0042] 2. Construction of an immortalized eggshell gland epithelial cell line SV40 overexpressing lentivirus carrying the Puromycin resistance gene marker, vector map as follows: Figure 2 The structure of the SV40 overexpression lentivirus is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with the sequence SEQ ID No:1 between the EcoRI and XbaI restriction sites of the starting vector pLVX-EF1α-IRES-puro, while keeping the other sequences of the vector pLVX-EF1α-IRES-puro unchanged.

[0043] 1) SV40 overexpression lentivirus transfection The eggshell gland epithelial cells obtained in step 1 were seeded into 6-well plates, with approximately 1 × 10⁶ cells per well. 5 After the cells adhere to the culture plate, change the medium. Add 1 mL of complete culture medium, then add 20 μL of SV40 overexpression lentivirus, mix well, and continue culturing. Observe the cell status after 12 hours and replace with fresh culture medium. When the cells have confluently grown, passage them into T25 culture flasks.

[0044] Transfection results as follows Figure 3 As shown, the cell morphology is clear and intact. After transfection, the cells are expanded for 3-4 generations before selection.

[0045] 2) Determination of the killing curve of puromycin Untransfected cells were packed at 5 × 10⁶ cells per well. 4 Cells were seeded into 24-well plates and incubated overnight. The next day, the old culture medium was removed from the 24-well plates. Fresh culture medium containing different concentrations of puromycin (1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL) was added to the seeded 24-well plates. The selection medium was replaced with fresh medium every two days, and cell viability was observed daily. The minimum concentration of puromycin used was the lowest concentration that killed all cells within 1-4 days. Based on the results, the optimal concentration of puromycin was determined to be 1 μg / mL, with an incubation period of 2 days.

[0046] 3) Puromycin screening of transfected cells On day 1, transfected cells were prepared at a ratio of 5 × 10⁶ cells per well. 4The cells were seeded into 24-well plates and incubated overnight. The next day, the old culture medium was removed from the 24-well plates. Selection medium containing 1 μg / mL puromycin was added, and the cells were incubated. The selection medium was replaced with fresh medium every two days, and the cell survival rate was observed daily. Cells surviving at the same time point were considered successfully transfected eggshell gland epithelial cells. The selected cells were then cultured. Figure 4 To expand the cells to the 15th generation, the cells have normal morphology and structure and are in good growth condition, and can be passaged in 2-3 days.

[0047] 3. Identification of immortalized cells 1) Immunofluorescence identification The immunofluorescence identification method is the same as described in step 1, and the results are as follows: Figure 5 As shown, the positive expression rate of CK18 protein was 97%, indicating that the cells were of epithelial origin.

[0048] 2) SV40 Integrated Detection DNA was extracted from primary eggshell gland epithelial cells and immortalized cells according to the instructions of the animal tissue / cell genomic DNA extraction kit.

[0049] Then, PCR amplification was performed using the SV40 Tag forward primer 5'-GACCTGTTGGTTGCACTGTG-3' and the reverse primer 5'-TGGTCTGAGTCATTGCTGCT-3'.

[0050] The reaction mixture (25 μL) consisted of: 100-200 ng template DNA, 0.5 μL each of primers (10 μM), 12.5 μL 2×PCR MasterMix, and ddH2O to a final volume of 25 μL. The PCR program was: 95℃ for 5 min, 35 cycles (95℃ for 30 s, 55-60℃ for 30 s, 72℃ for 30 s), 72℃ for 5 min, and storage at 4℃. A 1% agarose gel was prepared, and after loading the sample, electrophoresis was performed at 100V for 20-30 min. Finally, the gel imaging was observed.

[0051] The results are as follows Figure 6 As shown, the immortalized cell line iCUECs2025 exhibited a specific band, while primary cells showed no band, demonstrating that the immortalized eggshell gland epithelial cells expressed SV40, indicating that the eggshell gland epithelial cell line was successfully established.

[0052] The immortalized chicken uterine epithelial cell line iCUECs2025 was deposited at the China General Microbiological Culture Collection Center on November 4, 2025, with accession number CGMCC No. 46710.

[0053] Example 2: Construction of an Inflammation Model of Immortalized Eggshell Glandular Epithelial Cells Immortalized eggshell gland epithelial cells in the logarithmic growth phase were digested and resuspended to a concentration of 5 × 10⁻⁶. 4 Cell suspensions of cells / mL were seeded into 96-well plates. After incubation at 37°C and 5% CO2 for 24 hours, approximately 75% of cells adhered. The LPS stock solution was then diluted to a working concentration of 10 µg / mL with complete culture medium.

[0054] Remove the old culture medium. Add 100 µL of medium containing 10 µg / mL LPS to each well in the experimental group, and add an equal volume of complete culture medium to the control group. After culturing for 8 hours, remove the cells. Detect cell viability and nitric oxide content according to the nitric oxide kit instructions.

[0055] The results are as follows Figure 7 As shown, compared with the control group, 10 µg / mL LPS significantly reduced the activity of immortalized chicken shell gland epithelial cells, indicating that some epithelial cells had undergone apoptosis. Furthermore, nitric oxide is a typical marker of cellular inflammation; the significantly increased nitric oxide level in the LPS group indicates that the shell gland epithelial cell inflammation model was successfully established.

[0056] This invention successfully constructed an immortalized eggshell gland epithelial cell line. This cell line can be stably passaged multiple times, and its cell morphology and functional activity are well maintained. It can be used to construct an eggshell gland inflammation-related cell model.

[0057] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for constructing an immortalized eggshell gland epithelial cell line, characterized in that, It includes the following steps: 1) Obtaining SV40 overexpression lentivirus; 2) Transfect the lentivirus obtained in step 1) into primary chicken shell gland epithelial cells; after the transfected cells are expanded for 3-4 generations, they are screened for successful transfection with puromycin to obtain shell gland epithelial cells. 3) Passage the successfully transfected eggshell gland epithelial cells obtained in step 2) to obtain stable, immortalized eggshell gland epithelial cells.

2. The method according to claim 1, characterized in that, Step 1) describes an SV40 overexpression lentivirus containing a DNA molecule with the nucleotide sequence SEQ ID No:

1.

3. The method according to claim 1 or 2, characterized in that, In step 2), the puromycin screening concentration is 1 μg / mL, and the treatment time is 2 days.

4. The method according to claim 1 or 2, characterized in that, Step 3) describes a subculture of 15 generations.

5. A method for constructing an inflammatory model of immortalized eggshell gland epithelial cells, characterized in that, The inflammation model was obtained by culturing the immortalized eggshell gland epithelial cells according to any one of claims 1-4 in a medium containing LPS.

6. The method according to claim 5, characterized in that, The concentration of LPS used is 10 µg / mL.

7. The use of the method according to any one of claims 1-4 in the preparation of immortalized eggshell gland epithelial cell lines.

8. The use of the method according to any one of claims 1-4 in the preparation of products containing immortalized eggshell gland epithelial cell lines.

9. The chicken uterine epithelial immortalized cell line obtained by the method of any one of claims 1-4.

10. The use of the immortalized eggshell gland epithelial cells of claim 9 in studying the functions related to the eggshell gland in poultry and / or screening novel feed additives to improve eggshell quality.

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